Studies on Transglycosidation to Vitamin B6 by Microorganisms

Some properties of pyridoxine glucoside-synthesizing enzyme were studied using the partially and highly purified enzyme preparations from Micrococcus sp. No. 431.

The enzyme was stable at pH 7.0 and between 0°C and 30°C. The maximal activity was obtained at pH 8.0 and 37°C. Besides sucrose, phenyl-α-d-glucoside and maltose served as glucosyl donor. Of vitamin B₆ compounds tested, only pyridoxine served as glucosyl acceptor. The enzyme activity was inhibited by PCMB and heavy metal ions, and the inhibition was prevented by 2-mercaptoethanol, indicating the enzyme would be a sulfhydryl enzyme. The activity was not affected by chelating agents and not activated by metal ions.     

Acid-stable α-Amylase of Black Aspergilli

Some general properties of the acid-stable dextrinizing amylase of black Aspergillus were investigated comparing with those of Taka-amylase A. The mode of action on starch of this amylase was quite similar to that of Taka-amylase A. Saccharifying degree at red point in starch-iodine color reaction was 5.1% and the limit of starch saccharification was a little over 40 per cent calculated as glucose with both amylases. Maltase activity was absent. Degradation products in the course of starch hydrolysis were also quite similar and they mutarotated downward. So this amylase was decided to be α-type. Thermal stability of the acid-stable α-amylase was higher than that of Taka-amylase A. Its acid stability was much higher than that of Taka-amylase A. Taka-amylase A was inactivated completely at pH 2.2, 37°C, for 30 min, but the acid-stable α-amylase retained 87% of its original activity.

From the amylase preparation of black Aspergillus acid-stable α-amylase and acidunstable α-amylase were separated by gel filtration on sephadex G-100 column. From the acid-unstable α-amylase fraction this enzyme was purified by fractionations with rivanol and acetone, and finally obtained as a homogeneous protein after gel filtration with sephadex G-50. Comparison of some general properties between the two α-amylases was carried out. Catalytic action was quite similar with both enzymes, but dextrinizing unit per mg enzyme protein of the acid-unstable α-amylase was about 5.6 times as large as that of the acid-stable α-amylase. The acid-unstable α-amylase was less heat-stable than the acid-stable α-amylase. Acid stability and pH-activity curve were compared with both α-amylases. High stability of the acid-stable α-amylase in acidic condition was observed, but, in alkaline range, it was more sensitive than the acid-unstable α-amylase.     

Studies on Vitamin B6 Metabolism in Microorganisms

Escherichia freundii alkaline phosphatase was found in a membrane fraction and was purified by procedures involving spheroplast formation with lysozyme and EDTA, and DEAE-cellulose and Sephadex G-150 column chromatographies. Then this enzyme along with other phosphatases was investigated on the ability to transfer the phosphoryl group from p-nitrophenyl phosphate to pyridoxine. It was found that the ability of the transphosphorylation varied with these phosphatases. The transphosphorylation to hydroxy compounds such as alcohols, sugars and nucleosides was also compared. Escherichia freundii acid phosphatase showed the highest activity of transphosphorylation among phosphatases tested. The mechanism of transphosphorylation was discussed.

An enzyme, pyridoxamine 5′-phosphate transaminase, was purified from the cell-free extract of Clostridium kainantoi. The purification procedures involved ammonium sulfate fractionation, protamine sulfate treatment and, DEAE-cellulose, hydroxylapatite, DEAE-Sephadex and Sephadex G-200 column chromatographies. The purified enzyme, which had approximately 2700-fold higher specific activity over the original extract, showed a single schlieren pattern in the ultracentrifuge. From the spectral analysis, it seemed that pyridoxamine 5′-phosphate transaminase did not contain pyridoxal 5′-phosphate as a prosthetic group. It was recognized that the transamination was accelerated by the addition of amino acid and was inhibited by diisopropyl phosphofluoride. Glutamic acid formed in the reaction was identified to be a D-isomer. A study on the substrate specificity showed that the enzyme might be possible to be specific for pyridoxamine 5′-phosphate.

The extracellular formation of vitamin B₆ was searched in marine and terrestrial microorganisms. Two bacterial strains were selected and were identified as Vibrio and Flavobacterium, respectively. Marine microorganisms showed the considerable formation of vitamin B₆ and the presence of vitamin B₆ in sea water was also recognized. The cultural and reaction conditions for vitamin B₆ formation by these strains were investigated. Glycerol was commonly the most effective compound on vitamin B₆ formation among the compounds tested. It was suggested that both bacteria did not have the control system on vitamin B₆ biosynthesis by the amount of possible end products.     

Studies on the Metabolism of Pantothenic Acid in Microorganisms

The ability of the formation of coenzyme A from pantothenic acid and cysteine in the presence of AMP or ATP was searched in yeasts and bacteria. The result of screening showed that the activity was found in several yeasts and the bacteria belonging to the genera Sarcina, Corynebacterium and Brevibacterium. Particularly, Brevibacterium ammoniagenes IFO 12071 (ATCC 6871) accumulated a large amount of coenzyme A.

Isolation of the reaction products, which were synthesized by Brevibacterium ammoniagenes IFO 12071, were carried out. The isolates were identified as coenzyme A, dephosphocoenzyme A and phosphopantothenic acid.

The possibility for the formation of coenzyme A in a larger amount from pantothenic acid and cysteine was investigated with baker’s yeast under the condition coupled with ATP-generating system.

Effect of various factors affecting the accumulation of coenzyme A was investigated. Among them, glucose concentration and inorganic phosphorus concentration were the most important factors for its accumulation. Coenzyme A was not accumulated without the phosphorylation of AMP to ATP. Several cationic surfactants stimulated the accumulation of coenzyme A.

The amount of coenzyme A accumulated reached about 200 μg per ml of the reaction mixture under the suitable reaction conditions employed.     

Distribution of 7-Keto-8-aminopelargonic Acid Synthetase in Bacteria and the Control Mechanism of the Enzyme Activity

The 7-keto-8-aminopelargonic acid (KAPA) synthetase activities of cell-free extracts from various bacteria were investigated. The experiments on the substrate specificity of KAPA synthetase, using crude cell-free extracts from bacteria having high enzyme activity, showed that l-serine and pyruvic acid could replace l-alanine, but that, when the enzyme was partially purified, these compounds were not effective. Many kinds of amino acids such as l-cysteine, l-serine, d-alanine, glycine, d-histidine, and l-histidine, inhibited the enzyme activity. This inhibition was found to be competitive with l-alanine. Pyridoxal 5′-phosphate, which is a cofactor of the enzyme, also inhibited the enzyme activity at high concentrations. The repression of KAPA synthetase by biotin occurred in Bacillus subtilis and B. sphaericus but not in Micrococcus roseus and Pseudomonas fluorescens, even at a concentration of 1000 mµg per ml of biotin.     

Studies on the Utilization of Hydrocarbons by Microorganisms

Taxonomical studies on ten strains of hydrocarbon-utilizing bacteria reported in previous paper, which produced various kinds of amino acid, were carried out. They were Achromo-bacter cycloclastes, Achromobacter delmarvae, Bacillus species, Corynebacterium species, Micrococcus species. Many of them were not identical with the species which are described in Bergey’s Manual of 7th Edition.     

Fatty Acid Metabolism in Microorganisms

The production of pimelic acid from azelaic acid by microorganisms was studied. About 100 strains of bacteria which were able to utilize azelaic acid as a sole carbon source were isolated from soil and other natural materials. Among these bacteria, several strains produced a large quantity of an organic acid (pimelic acid) from azelaic acid in their culture fluids during the cultivation. The acid was isolated from the culture fluid of strain A133 in crystalline form. The crystal was identified as pimelic acid by physicochemical and biological methods.

From the results of investigations on the morphological and physiological characters, the bacterial strain A133 was assumed to be Micrococcus sp.     

Lipase from Candida paralipolytica

The extracellular lipase from Candida paralipolytica required essential activators* (usually bile- or calcium salts) for the in vitro hydrolysis of triglycerides. The reaction systems emulsified with gum arabic, gelatin, lecithin, methyl cellulose, pectin, polyvinyl alcohol, sodium cholate, or without emulsifier were compared concerning requirement for essential activator, inhibition with sodium chloride and maximum reaction rate, and the following findings have been obtained. (1) The emulsions used can be classified into five groups by the essential activator requirement. (2) The inhibition with sodium chloride depended on reaction system. (3) Each reaction system gave a similar reaction rate at pH 8.2. (4) Long-chain fatty acid dissolved in substrate was necessary to the activation with calcium salts.     

Lipids and Related Substances Inducing the Lipase Production by Candida paralipolytica

Properties of autolytic breakdown of rat skeletal muscle proteins in the alkaline pH range have been reported. The activity is almost exclusively localized in the myofibrillar fraction, but is not solubilized with Triton X-100. The activity is affected by the KCI concentration in the reaction mixture. In 0.6 M and the more concentrated KCI solutions, the maximum activity is attained. The optimum pH of the activity is in the range of pH 7.5～9.5, and the optimum temperature is between 47～57°C.

This autolytic activity seems to be different from catheptic activity which shows its optimum pH in the acid pH range. Moreover, though more than half of the catheptic activity of rat skeletal muscle is recovered in the myofibrillar fraction, the catheptic activity in the myofibrillar fraction can be removed from the fraction by the extraction with dilute saline solution containing Triton X-100.